1Leverette R, 1Keyser B, 2Seymour A and 2Hollings M
1Scientific & Regulatory Affairs, RAI Services Company, Winston-Salem, NC 27101
2Covance Laboratories Ltd, Otley Road, Harrogate, North Yorkshire HG3 1PY, UK
The preferred means of assessing combustible or other aerosol-generating tobacco products in vitro is by direct exposure of cell cultures to freshly generated whole mainstream smoke/aerosols. This approach eliminates the fractionation of smoke/aerosols that occurs when collecting particulate matter on filter pads and gas vapor phase via liquid traps. Whole smoke/aerosol exposure systems are commercially available and have been utilized to assess combustible and tobacco heating products (THP) as well as electronic nicotine delivery systems (ENDS) in vitro. However, a challenge with such systems is ensuring a sufficient number of doses and sample throughput for in vitro toxicological studies in a timely manner. Vitrocell® has developed a high throughput whole smoke/aerosol exposure module (Ames 48 module) designed to concurrently deliver up to seven different doses (six wells per dose) of smoke/aerosol and a clean air control to 48 wells of bacterial cell cultures. Characterization of smoke/aerosol delivery within this system was conducted in a series of experiments designed to assess smoke/aerosol delivery and biological responses from a Kentucky Reference 3R4F combustible cigarette or a commercially available THP or ENDS. Dilution airflows consisting of 0.5 – 10 L/min for 3R4F and 0 (undiluted) - 4 L/min for the THP and ENDS were evaluated. Smoke/aerosol deposition was quantified on a mass delivered basis using fluorescence measurements (Ex 355/Em 485) of captured particulate matter and chemical analysis (e.g., glycerol, nicotine) of either DMSO (3R4F) or PBS (THP, ENDS) liquid traps within the module. The mutagenicity (Ames Assay) of whole smoke from the 3R4F cigarette was also assessed with the AMES 48 module using Salmonella strains TA98 and TA100 (±S9). Results demonstrate a dose-dependent deposition of smoke/aerosol constituents (3R4F, THP and ENDS) and a characteristic dose-dependent increase in revertant counts (3R4F). Current test results from the Ames 48 module are comparable to historical 3R4F results generated using the standard Vitrocell® exposure modules.